PROJECT SUMMARY Bacteria interact with their environments primarily through proteins attached to their surfaces. These proteins enable different lifestyles?and in the case of pathogens, direct the course of infection. The diverse mechanisms responsible for attaching surface proteins have been investigated extensively and are frequently used as targets in therapeutic intervention. A number of pathogenic bacteria produce infectious spores that display a variety of important surface proteins; however, little is known about how these proteins are attached. A prime example is the collagen-like protein BclA, which is attached to the surface of spores of Bacillus anthracis, the causative agent of anthrax. The outmost exosporium layer of B. anthracis spores is composed of an external hair-like nap and an underlying basal layer. The filaments of the nap are formed by trimers of BclA, which are tightly attached to basal layer protein BxpB. BclA attachment occurs through its 38-residue amino terminal domain (NTD), which is proteolytically processed during sporulation to remove residues 1-19. This event apparently triggers BclA attachment (through residues 20-38) to BxpB to form complexes that are resistant to conditions that routinely disrupt noncovalent interactions. The protease responsible for BclA processing has not been identified, but it does not appear to be BxpB or an enzymatically active domain of BclA. Recombinant versions of BclA and BxpB can form extremely tight complexes in vitro, suggesting that other factors are not required for attachment. The goal of this study is to define the stable interaction between BclA and BxpB. This bond is likely to be covalent. To identify this bond, BclA-BxpB complexes will be proteolytically digested, and the resulting peptides will be separated and sequenced by liquid chromatography-tandem mass spectrometry. The results will identify covalently linked residues and the chemical nature of the covalent bond. Additionally, BclA and BxpB binding sites will be mapped by using hydrogen deuterium exchange by mass spectrometry. The nature of these sites can suggest a mechanism for covalent bond formation and, in the event that a covalent bond is not involved, will identify noncovalent interactions capable of forming an unusually tight protein complex. The expected outcome of this study is the elucidation of an important and new type of protein attachment to a bacterial cell, a spore in this case. This mechanism is likely shared by many spore-forming bacteria that attach collagen-like proteins to their spores, including pathogens such as Clostridium difficile. It is also possible that non-spore-forming organisms use a mechanism analogous to that of BclA-BxpB attachment to form stable protein complexes. Furthermore, BclA and BclA homologs play important roles in pathogenesis and persistent attachment to environmental surfaces. Thus, understanding the mechanism of BclA attachment affords new targets for treatment and prevention of anthrax and other diseases.